The Production of D-2,3-Butanediol and Meso-2,3-Butanediol with High Optical Purity in Engineered Bacillus Licheniformis WX02

Abstract: Microbial production of 2,3-butanediol (2,3-BD) has received increasing interests because of its potential applications in cosmetics, foods, transport fuels, medicines, and polymers industries. Bacillus licheniformis WX-02 is a generally regarded as safe (GRAS) strain and is regarded as a potential 2,3-BD producer. However, this strain like other 2,3-BD producing microorganisms produces a mix of D-2,3-BD and meso-2,3-BD isomers. In order to reduce the cost of purifying a specific 2,3-BD isomer from fermentation broth, it is desirable for the microbial strain to produce the specific target isomer without synthesis of the other forms. In this study, the budC gene was identified responsible for the conversion of the precursor acetoin to meso-2,3-BD in B. licheniformis. A budC deletion strain of B. licheniformis WX-02 was successfully constructed to inhibit the flux of acetoin to meso-2,3-BD biosynthesis, and D-2,3-BD with high optical purity was successfully produced. Furthermore, glycerol dehydrogenase (Gdh) was identified as the catalyst in D-2,3-BD biosynthesis from acetoin in B. licheniformis. The gdh gene was therefore deleted from the wild-type strain to produce high purity meso-2,3-BD. In addition, the acoR gene involved in acetoin degradation was also deleted to increase the pool of the precurcor acetoin. The double deletion mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD isomer with > 99% purity in flask culture. Using fed-batch fermentation, the highest titer (98.0 g/L) ever reported for meso-2,3-BD was achieved by WX-02ΔgdhΔacoR.